Cellular and molecular imaging technologies are revolutionizing neuroscience. The technology with the biggest potential impact on this field is non-linear optical microscopy which enables two-photon (2P) imaging of fluorescent structures deep in living tissue with unprecedented spatial and temporal resolution. Combining 2P imaging with electrophysiology and molecular uncaging creates an extraordinarily powerful tool that is having a profound effect on the conduct of neuroscientific study. The strategic goal of this revised proposal is to put this critical technology in the hands of a two highly productive groups of neuroscientists at Northwestern University (NU) that receive nearly $8M/year (direct costs) in funding from NINDS. One of these groups is focused on the properties of neuronal dendrites in health, aging and disease; the other group is focused on neural stem cell biology and its application to neuroregeneration. To maximize the investment of NU and NINDS in these research programs, both groups need ready access to user-friendly, multifunctional 2P imaging workstations. The following specific aims are designed to achieve this goal: 1. to further characterize and optimize the performance of our existing 2P imaging workstations; 2. to extend the capabilities of the existing workstations by adding new hardware and software features integrating electrophysiology and molecular uncaging; 3. to create additional core facilities with 2P imaging workstations and technical support for NIH funded investigators who do not currently have access to these resources; 4. to establish an infrastructure that ensures the safe, efficient and productive operation of all the cores; 5. to create an Internet-based distribution point for dissemination of information about applications, software and hardware design/implementation A corporate partner with expertise in the production of 2P workstations, Prairie Technologies, has been recruited to help achieve these aims. By establishing a dialogue between industry and researchers, we hope to accelerate the development of hardware and software that best meets the needs of the neuroscience community. The attainment of these aims will not only have a transforming impact on the NIH funded research programs at NU but it will significantly accelerate the delivery of these technologies to the broader neuroscience community, quicken the pace of scientific discovery and promote the development of new treatments for neurological disorders.